This invention relates to carbohydrate metabolism in plants, and in particular to sucrose-binding proteins (SBPs). Aspects of the invention include a novel SBP gene isolated from soybean, and modified SBPs having enhanced sucrose uptake activity. Nucleic acid vectors, transgenic cells and transgenic plants having modified sucrose uptake activity are also provided. The invention also relates to promoter sequences useful for controlling expression of transgenes in plants, including SBP transgenes.
The regulation of sucrose transport in plants has a major impact on plant growth and productivity. Through photosynthesis, plants fix atmospheric carbon dioxide into triose phosphates, which are then used to produce sucrose and other carbohydrates. These carbohydrates are then transported throughout the plant for use as energy sources, carbon skeletons for biosynthesis and storage for future growth needs. Sucrose is the major form of transported carbohydrate. The ability of plant cells actively to transport sucrose across the plasma membrane so that the sucrose that is mobilized in the phloem can be taken into cells for use is a critical step in sucrose utilization.
The development of plant seeds involves the accumulation of carbon and nitrogen reserves in forms that can both withstand desiccation and be utilized as an energy source by the developing embryo during germination. The accumulation of carbon in developing seeds is mediated by specific plasma membrane proteins (Overvoorde et al., 1996; Riesmeier et al., 1992; Bush, 1993). Photoaffinity labeling of membranes isolated from soybean cotyledon tissue with a photolyzable sucrose analog identified a distinct 62 kD sucrose-binding protein, or SBP (Ripp et al., 1988). Analysis of the cDNA encoding the SBP and its deduced amino acid sequence indicates that the SBP contains a single hydrophobic domain at its N-terminus but otherwise is a hydrophilic protein lacking the expected membrane-spanning hydrophobic segments typically present in transport proteins (Grimes et al., 1992). Biochemical analysis of the topology of the SBP demonstrates that it is tightly associated with the external leaflet of the plasma membrane (Overvorrde and Grimes, 1994). The involvement of the SBP in sucrose uptake was implicated by immunolocalization experiments demonstrating that the SBP is exclusively associated with the plasma membrane of cells involved in active sucrose uptake (Grimes et al., 1992). Kinetic analysis of SBPmediated sucrose uptake in a yeast system indicates that the uptake is specific for sucrose but is proton independent and relatively nonsaturable, thus defining a novel mechanism for sucrose uptake (Overvoorde et al., 1996).
Sucrose uptake in developing seeds affects two significant agricultural characteristics of the mature seed: the carbohydrate content of the resulting seed grain, and the vitality of the seedling that emerges when the seed grain is planted. Enhanced sucrose uptake activity in developing seeds may be desirable where it is an advantage to increase the carbohydrate content of the seed (e.g., where the seed is the primary plant material harvested, such as soybean). In contrast, decreased sucrose uptake activity in seeds might be desirable where the vegetative material of the plant is harvested. Thus, plants having modified sucrose uptake activity during seed development would be of significant agricultural importance, and it is to such plants that the present invention is directed.
The present invention provides isolated nucleic acid molecules encoding plant sucrose binding proteins, which are key proteins in the uptake of sucrose into developing seeds. In one embodiment, the invention provides modified forms of sucrose binding proteins that are shown to have enhanced sucrose uptake activity.
The previously described sucrose binding protein from soybean (Overvoode et al., 1996) is herein referred to as SBP1. A new SBP is provided herein and is referred to as SBP2. The SBP2 polypeptide is shown to be 489 amino acid residues in length, and to be expressed at enhanced levels during seed development. The SBP2 polypeptide is shown to have sucrose uptake activity in a heterologous yeast assay system.
In addition, modified forms of the SBP1 and SBP2 proteins are provided having enhanced sucrose uptake activity. In one embodiment, such forms are deletion mutants in which amino acid residues are removed from the C-terminus of the proteins. By way of example, removal of 80 amino acid residues from the C-terminus of the SBP1 protein is shown to produce increased sucrose uptake in the yeast assay system.
The invention also provides 5xe2x80x2 regulatory regions (including promoter sequences) of the soybean SBP1 and SBP2 genes. These regulatory regions confer specific or enhanced expression in developing seeds and so may be used to express any transgene in developing seeds.
Thus, in one aspect, the invention provides a modified plant sucrose binding protein wherein the modified sucrose binding protein has a modified amino acid sequence compared to a corresponding wild-type sucrose binding protein, and wherein expression of the modified sucrose binding protein in a yeast assay system confers enhanced sucrose uptake compared to the corresponding wild-type sucrose binding protein. In particular embodiments, modified sucrose binding proteins provided by the invention enhance sucrose uptake in the yeast assay system by at least 10%, and preferably by at least 25%, compared to the wild-type sucrose binding protein. In certain embodiments, the modified plant sucrose binding proteins have a modified amino acid sequence comprising a C-terminal truncation compared to the wild-type sucrose binding protein. Such a truncation is typically of between about 10 and about 100 amino acids, and is preferably of about 80 amino acids. Although such modified SBPs may be produced from any known sucrose binding proteins, modified forms of SBP1 and SBP2 are exemplary of the invention. Modified forms of SBP1 and SBP2 include those forms having the amino acid sequences shown in Seq. I.D. Nos. 2 and 4, respectively.
In another aspect of the invention, nucleic acid molecules encoding modified plant sucrose binding proteins are provided, together with vectors comprising such nucleic acid molecules. The invention also provides transgenic plants expressing modified sucrose binding proteins. Such transgenic plants may have modified sucrose uptake activity, particularly in developing seeds.
In another aspect, the invention provides an isolated nucleic acid molecule encoding a SBP2 sucrose binding protein or a variant of a SBP2 protein. Such proteins may comprise an amino acid sequence as shown in Seq. I.D. Nos. 3 and 4, or sequences having at least 70% and preferably at least 90% sequence identity with these sequences. Recombinant expression cassettes comprising such nucleic acid molecules are also provided by the invention, as are transgenic plants comprising such recombinant expression cassettes.
Another aspect of the invention is a recombinant nucleic acid molecule comprising a promoter sequence operably linked to a nucleic acid sequence, wherein the promoter sequence comprises a SBP1 or SBP2 promoter. Such promoters preferably comprise at least 25 consecutive nucleotides of the 5xe2x80x2 regulatory sequences shown in Seq. I.D. Nos. 6 and 7. In particular embodiments, the nucleic acid sequence comprises a plant sucrose binding protein. Transgenic plants comprising such recombinant nucleic acid molecules are also an aspect of the invention.
These and other aspects of the invention are discussed in more detail in the following description.